Proteomic characterization of the human centrosome by protein correlation profiling

Abstract

The centrosome is the major microtubule-organizing centre of animal cells and through its influence on the cytoskeleton is involved in cell shape, polarity and motility. It also has a crucial function in cell division because it determines the poles of the mitotic spindle that segregates duplicated chromosomes between dividing cells. Despite the importance of this organelle to cell biology and more than 100 years of study, many aspects of its function remain enigmatic and its structure and composition are still largely unknown. We performed a mass-spectrometry-based proteomic analysis of human centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions. True centrosomal proteins were revealed by both correlation with already known centrosomal proteins and in vivo localization. We identified and validated 23 novel components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins. Protein correlation profiling permits the analysis of any multiprotein complex that can be enriched by fractionation but not purified to homogeneity.

Interference of the dominant negative helix–loop–helix protein ID1 with the proteasomal subunit S5A causes centrosomal abnormalities

“…Analysis of the subcellular localization of endogenous S5A protein revealed that, similar to ID1, a fraction of cellular S5A is localized at centrosomes. Similar centrosomal localization patterns have been reported for other components of the proteasome (Freed et al, 1999;Wigley et al, 1999;Fabunmi et al, 2000;Andersen et al, 2003), Localization of proteasome components at the centrosome might be crucial for centrosome duplication (reviewed in Hinchcliffe and Sluder, 2001). Supporting an important function of proteasome components during centrosome duplication are our findings that knockdown of S5A by shRNA caused an increase of cells with supernumerary centrioles (Figure 4).…”

Antizyme, a mediator of ubiquitin-independent proteasomal degradation and its inhibitor localize to centrosomes and modulate centriole amplification

“…To date, partial centrosome localization has been shown for several proteins involved in ubiquitination and proteasomal degradation (Fabunmi et al, 2000;Andersen et al, 2003). We now show that a fraction of antizyme 1 associates with maternal centrioles and modulates daughter centriole amplification.…”

“…Recent evidence has shown that many components of the proteasomal degradation machinery concentrate at centrosomes and form active proteasomal complexes (Fabunmi et al, 2000;Andersen et al, 2003). We show here that antizyme 1, a mediator of ubiquitin-independent proteasomal degradation and its inhibitor, AZI, localize to centrosomes and are functionally important centrosome-associated proteins.…”

An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

“…Andersen et al originally identified CEP63 in a proteomic analysis of the human centrosome and confirmed centrosomal localisation by expressing GFP construct in cells (Andersen et al, 2003). At the time we isolated XCEP63, no CEP63 characterization studies had been published and consequently no function had been ascribed.…”